Interaction of endotoxic lipid A and lipid X with purified human platelet protein kinase C.
Lipid A, the toxic principle of endotoxic lipopolysaccharide, and its precursor, Lipid X, interact with human platelets and modulate protein kinase C therein (Grabarek, J., Timmons, S., and Hawiger, J. (1988) J. Clin. Invest. 82, 964-971). We have now purified protein kinase C from human platelets and studied its interaction with endotoxic Lipids A and X. Protein kinase C-dependent phosphorylation of histone III-S was increased 15 times in the presence of Lipid A and 300 microM Ca2+. The Ca2+ requirement for such activation was lower when 4 beta-phorbol 12-myristate 13-acetate (PMA) or 1,2-diolein were added. Lipid A also induced autophosphorylation of protein kinase C, and its activation was enhanced by phosphatidylserine without reducing the Ca2+ requirement. Kinetic analysis of protein kinase C activation induced by Lipid A, in regard to ATP as a substrate, demonstrated that Lipid A increased the rate of the reaction (Vmax) without modifying the affinity of the enzyme (Km) for the substrate. Lipid X inhibited the activation of the enzyme induced by Lipid A. Lipid X also inhibited protein kinase C activation by phosphatidylserine, 1,2-diolein, and PMA. However, 10 times more of Lipid X was required for 50% inhibition (IC50) when PMA was used as an activator of protein kinase C in the presence of phosphatidylserine than when Lipid A and 1,2-diolein were used. These results support the hypothesis that endotoxic Lipid A and Lipid X exert their biological effect in platelets through direct interactions with protein kinase C.
Romano
M
M
Department of Medicine, New England Deaconess Hospital, Boston, Massachusetts.
Hawiger
J
J
D001792:Blood Platelets / Q000201:enzymology*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002474:Cell-Free System; D004731:Endotoxins / Q000378:metabolism*; D004789:Enzyme Activation; D006017:Glycolipids / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007526:Isoelectric Point; D007700:Kinetics; D008050:Lipid A / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*
D004731:Endotoxins; D006017:Glycolipids; D008050:Lipid A; C044283:lipid X; D011493:Protein Kinase C; D002118:Calcium